1. Treatment with ipilimumab
Table S1
Treatment with ipilimumab
Responder
Non-responder
No. Patients 30 48
Number of received doses (ipilimumab 3 mg/kg)
4 doses 22 24
3 doses 4 5
2 doses 3 7
1 dose 6
Unknown 1
Table S1: Patients received ipilimumab 3 mg/kg within clinical trials and later on as approved therapy. From 78 treated patients 46 patients received four doses, nine patients received three doses, ten patients received two doses and 6 patients received one dose. For 7 patients the number of received doses is unknown. The number of infusions was similar in both groups. Patients did not receive all four doses due to side effects or progression of disease.

2. Figure S1: Pretreatment of patients in stage IV before administration of ipilimumab. More than 48% (38 out of 78 patients) received at least three prior treatment regimens before undergoing therapy with ipilimumab.

3. Specimen-Nr.
Tumor content (% area)
PD-L1 expression (40-delta CT)
Expression classification (cut-off at CT 28,86)
% tumor membrane expression of PD-L1
IHC classification (positive=>5% stained tumor cells
IHC staining (P=Princeton clone 28-8; E=Erlangen, GTX117446) 1 40
29,93
PD-L1 low
INDETERMINATE* NE P 2 73
24,98
negative 3 50
27,11 4 35
28,78 5 75
29,05
PD-L1 high 6 25
29,33 7 90
29,42 8 65
29,90 9
30,20 10 60
30,25 11
31,33
positive 12 98
31,41 13 20
32,04 14 95
27,90 E 15
27,80 16
28,60 17
29,10 18 19 85
30,78
31,01 21
31,05 22
31,11 23
31,16 24
31,20
31,25 26 27 70
31,30 28
31,31 29 30 31
31,53 32
31,54 33 80
31,68 34
31,70 36
32,17 37
100
32,74 38 55
32,76
* due to high melanin content
Figure S2: Immunohistochemical staining of 38 specimen for PD-L1 expression. Specimen with > 5% membranous staining were categorized “positive”. Shaded in green: Concordance between RT-PCR and IHC. NE: Not evaluable due to too much melanin

4. Concordance between RT-PCR and immunohistochemistry
PD-L1 antibody
Positive concordance between RT-PCR and immunohistochemical staining
No concordance between RT-PCR and immunohistochemical staining
BMS/Dako antibody (Princeton)
36 %
64 %
CD274 antibody (Erlangen)
80 %
20 %
Figure S3: Correlation of expression data from RT-PCR with immunohistochemical staining of specimen for PD-L1 expression in 38 cases. The BMS/DAKO antibody (Princeton) did not deliver any result in 2 out of 13 cases due to too much melanin and concordance with RT-PCR classification in only 4/11 cases. The CD274 antibody (Erlangen) showed better correlation with the RT-PCR results with concordance in 20/25 cases.